Hereditary Dentin Defects (HDD) affect 1 in 8,000 people. The genetic causes of most HDD correlate with the dysfunction of dentin proteins: type I collagen and dentin sialophosphoprotein (DSPP). All DSPP mutations reported to date show a dominant pattern of inheritance. This is because DSPP mutations manifest their phenotype through a dominant negative or gain of function mechanism?not by haplo- insufficiency. Reducing the normal amount of DSPP by half, as in Dspp heterozygous mice, does not cause dentin malformations. Dspp-/- null mice show a severe phenotype due to the absence of DSPP?not by the autosomal dominant pathological mechanism that causes HDD in humans. This distinction is important. Therapeutically, HDD in the absence of Dspp-/- could be reversed by restoring DSPP expression, whereas human HDD caused by DSPP mutations could not be restored in this way because the condition is not due to a lack of DSPP protein, but rather, is due to the pathological effects of aberrant DSPP in odontoblasts. This proposal ?DSPP Function, Pathophysiology, and Genetic Diagnosis? seeks to improve our under- standings of 1) DSPP-derived proteins during normal dentinogenesis, 2) the pathological mechanism of Dspp -1 frameshift mutations, and 3) to develop a practical approach for HDD genetic testing to specifically identify the causative mutation and establish a definitive diagnosis. Three Specific Aims are proposed: SA1: Determine the role of DSPP-derived proteins during initial dentin mineral formation and coalescence by characterizing early dentin mineralization in Dspp+/+, Dspp-1fs/-1fs, Dspp-2fs/-2fs and Dspp-/- mice. SA2: Localize the DSPP -1 frameshift protein in vivo to determine where it accumulates and causes odontoblast cell pathology. SA3: Improve the diagnosis and management of HDDs by establishing an efficient genetic testing algorithm (sequence of actions that identifies the exact genetic cause of HDD in a given individual). Strategy: We hypothesize that DSPP helps initiate the mineralization of dentin calcospherites and promotes their growth and coalescence into a continuous mineral layer. By characterizing and comparing early dentin mineralization in Dspp+/+, Dspp-/-, and Dspp-2fs/-2fs mice using Focus Ion Beam Scanning Electron Microscopy (FIB-SEM), we can determine if dentin sialoprotein (DSP) or dentin phosphoprotein (DPP) is promoting the initiation and/or coalescence of dentin. We hypothesize that DSPP -1 frameshift mutations cause odontoblast cell pathology, possibly through ER stress. We test this hypothesis using Dspp -1 frameshift knockin mice that closely mimic human disease. Odontoblast pathology is assessed by FIB-SEM and TEM double immunogold labeling for the mutant protein and organelle markers in vivo. To improve the diagnosis and management of HDD, we apply a genetic testing algorithm to recruited HDD families to optimize its reproducibility and efficiency in identifying the underlying disease-causing mutations.